Methods for preparing purified lipopeptides

ABSTRACT

The present invention relates to crystalline and crystal-like forms of lipopeptides, including daptomycin, a lipopeptide antibiotic with potent bactericidal activity against gram-positive bacteria, including strains that are resistant to conventional antibiotics. The present invention relates to methods of purifying lipopeptides, including daptomycin, a lipopeptide antibiotic with potent bactericidal activity against gram-positive bacteria, including strains that are resistant to conventional antibiotics. The present invention also relates to pharmaceutical compositions comprising the purified form of the lipopeptide and methods of using these compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/955,495, which was filed on Jul. 31, 2013 and issued as U.S.Pat. No. 8,846,610 on Sep. 30, 2014; which is a continuation of U.S.patent application Ser. No. 12/198,666, which was filed Aug. 26, 2008and issued as U.S. Pat. No. 8,697,638 on Apr. 15, 2014; which is acontinuation of U.S. patent application Ser. No. 11/108,380, filed Apr.18, 2005; which is a continuation-in-part application of U.S. patentapplication Ser. No. 10/023,517, filed Dec. 17, 2001, which claims thebenefit of U.S. Provisional Application No. 60/256,268, filed Dec. 18,2000, U.S. Provisional Application No. 60/274,741, filed Mar. 9, 2001,U.S. Provisional Application No. 60/341,315, filed Dec. 13, 2001, andU.S. Provisional Application No. 60/340,525, filed Dec. 13, 2001. U.S.patent application Ser. No. 11/108,380 is also a continuation-in-part ofU.S. patent application Ser. No. 10/024,701, filed Dec. 17, 2001, whichclaims the benefit of U.S. Provisional Application No. 60/256,268, filedDec. 18, 2000, U.S. Provisional Application No. 60/274,741, filed Mar.9, 2001, U.S. Provisional Application No. 60/341,315, filed Dec. 13,2001, and U.S. Provisional Application No. 60/340,525, filed Dec. 13,2001. U.S. application Ser. No. 11/108,380 is also acontinuation-in-part of U.S. patent application Ser. No. 10/024,405,filed Dec. 18, 2001, which claims the benefit of U.S. ProvisionalApplication No. 60/256,268, filed Dec. 18, 2000 and U.S. ProvisionalApplication No. 60/274,741, filed Mar. 9, 2001. The contents of each ofthe above-referenced applications are incorporated herein by referencein their entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to crystalline and crystalline-like formsof lipopeptides, including daptomycin, a lipopeptide antibiotic withpotent bactericidal activity against gram-positive bacteria, includingstrains that are resistant to conventional antibiotics. The presentinvention also relates to processes for preparing crystalline orcrystal-like forms of the lipopeptide and to methods of purifyinglipopeptides including daptomycin. The present invention also relates topharmaceutical compositions comprising the purified form of thelipopeptide and methods of using these compositions.

BACKGROUND OF THE INVENTION

The rapid increase in the incidence of gram-positiveinfections—including those caused by antibiotic-resistant bacteria—hassparked renewed interest in the development of novel classes ofantibiotics. One such class is the lipopeptide antibiotics, whichincludes daptomycin. Daptomycin has potent bactericidal activity invitro against clinically relevant gram-positive bacteria that causeserious and life-threatening diseases. These bacteria include, but arenot limited to, resistant pathogens, such as vancomycin-resistantenterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA),glycopeptide intermediary susceptible Staphylococcus aureus (GISA),coagulase-negative staphylococci (CNS), and penicillin-resistantStreptococcus pneumoniae (PRSP), for which there are very fewtherapeutic alternatives. See, e.g., Tally et al., 1999, Exp. Opin.Invest. Drugs 8:1223-1238.

Daptomycin's inhibitory effect is a rapid, concentration-dependentbactericidal effect in vitro and in vivo, and a relatively prolongedconcentration-dependent post-antibiotic effect in vivo.

Daptomycin is described by Baltz in Biotechnology of Antibiotics, 2ndEd., ed. W. R. Strohl (New York: Marcel Dekker, Inc.), 1997, pp.415-435. Daptomycin, also known as LY 146032, is a cyclic lipopeptideantibiotic that can be derived from the fermentation of Streptomycesroseosporus. Daptomycin is a member of the factor A-21978C₀ typeantibiotics of S. roseosporus and is comprised of a decanoyl side chainlinked to the N-terminal tryptophan of a cyclic 13-amino acid peptide(FIG. 1). Daptomycin has an excellent profile of activity because it ishighly effective against most gram-positive bacteria; it is highlybactericidal and fast-acting; it has a low resistance rate and iseffective against antibiotic-resistant organisms. The compound iscurrently being developed in a variety of formulations to treat seriousinfections caused by bacteria, including, but not limited to,methicillin-resistant Staphylococcus aureus (MRSA) andvancomycin-resistant enterococci (VRE).

A number of United States patents describe A-21978C₀ antibiotics anddaptomycin-related lipopeptides including daptomycin (LY 146032). Thesepatents also describe methods of producing and isolating the A-21978C₀antibiotics and daptomycin-related lipopeptides.

U.S. Pat. Nos. RE32,333, RE32,455, 4,800,157, 4,874,843, and 4,885,243describe methods of synthesizing and isolating daptomycin fromfermentation cultures of Streptomyces roseosporus. U.S. Pat. Nos.RE32,310, RE32,311, 4,537,717, 4,482,487 and 4,524,135 describeA-21978C₀ antibiotics and methods of deacylating the A-21978C₀antibiotic and reacylating the peptide nucleus and antibioticderivatives made by this process. U.S. Pat. No. 5,912,226 (hereafter the'226 patent) describes the identification and isolation of twoimpurities produced during the manufacture of daptomycin,anhydro-daptomycin and the β-isomer form of daptomycin. None of theseUnited States patents discloses a method for precipitating orcrystallizing a lipopeptide in a manner to increase purity of thelipopeptide.

U.S. Pat. No. 4,439,425 (hereafter the '425 patent) discloses acrystalline lipopeptide and a method of crystallizing the lipopeptide.The lipopeptide disclosed in the '425 patent is structurally dissimilarfrom daptomycin and daptomycin-related lipopeptides. U.S. Pat. No.5,336,756 (hereafter the '756 patent) also discloses a crystallinecyclic lipopeptide comprising a hexapeptide. The crystalline cycliclipopeptide disclosed in the '756 patent is also structurally dissimilarfrom daptomycin and daptomycin-related lipopeptides. The '756 patentdiscloses that the lipopeptide, an echinocandin-type compound, can beobtained when aqueous n-propanol is employed as the crystallizingsolvent. See, e.g., cols. 1-2 of the '756 patent. Neither the '425patent nor the '756 patent disclose methods of crystallizing orprecipitating daptomycin or a daptomycin-related lipopeptide, nor dothey disclose methods of crystallizing or precipitating lipopeptidesproduced by Streptomyces.

It would be advantageous to develop a method of crystallizing orprecipitating daptomycin and daptomycin-related lipopeptides to providean improved purification method for these lipopeptides. In addition, acrystalline or highly purified precipitated form of daptomycin or otherdaptomycin-related lipopeptide would be useful in formulatingpharmaceutical compositions for treating bacterial infections. Further,a crystalline or highly purified precipitated form of daptomycin ordaptomycin-related lipopeptide would be useful in a method to make asterile product, particularly bulk sterile product. Thus, there is aneed for methods to produce crystalline or precipitated daptomycin anddaptomycin-related lipopeptides and the crystalline or precipitatedforms of the lipopeptides produced thereby. However, there has been nosimple and robust method that has been effective in crystallizing orprecipitating daptomycin or a daptomycin-related lipopeptide thatresults in a lipopeptide that is more pure after crystallization orprecipitation than before.

SUMMARY OF THE INVENTION

The instant invention addresses these problems by providing crystallineand crystalline-like forms of lipopeptides, particularly daptomycin anddaptomycin-related lipopeptides and methods for producing them. In oneembodiment, the invention provides methods for crystallizinglipopeptides. In another embodiment, the methods provide a lipopeptidethat is more pure after crystallization or precipitation than beforecrystallization or precipitation.

The invention also provides robust processes for producing and purifyinglipopeptides comprising, inter alia, crystallizing or precipitatinglipopeptides. In one embodiment, the crystallizing or precipitatingsteps of the processes are used to purify the lipopeptides. In anotherembodiment, the processes are used for large-scale and/or commercialproduction of lipopeptides, preferably daptomycin.

The invention further provides highly purified crystalline orcrystal-like forms of daptomycin and daptomycin-related lipopeptides. Inone embodiment, the crystalline or crystal-like forms of thelipopeptides may be used in pharmaceutical compositions. In anotherembodiment, the invention comprises methods of using the pharmaceuticalcompositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of daptomycin.

FIG. 2 shows a photomicrograph of urchin-like crystal or crystal-likeparticle of daptomycin produced by the method described in Example 12.

FIG. 3 shows a photomicrograph of needle-like crystals of daptomycin.

FIG. 4 shows a photomicrograph of rod-like crystals of daptomycin.

FIGS. 5A-F show photomicrographs of daptomycin samples at 100×magnification. FIG. 5A shows amorphous daptomycin using planetransmitted light. FIG. 5B shows amorphous daptomycin using crossedpolarized light. FIG. 5C shows daptomycin crystals using planetransmitted light. FIG. 5D shows daptomycin crystals using crossedpolarized light. FIG. 5E shows daptomycin crystals using planetransmitted light. FIG. 5F shows daptomycin crystals using crossedpolarized light. The daptomycin crystals were produced by the protocoldisclosed in Example 7.

FIG. 6 shows an x-ray powder diffraction pattern for amorphousdaptomycin.

FIG. 7 shows an x-ray powder diffraction pattern for a daptomycincrystal produced by the protocol described in Example 7.

FIG. 8 shows an x-ray powder diffraction pattern for a second sample ofa daptomycin crystal produced by the protocol described in Example 7.

FIG. 9 shows birefringence of a crystal-like particle of daptomycin whenexposed to polarized light. The crystal-like particle was produced bythe method described in Example 12.

FIG. 10 shows a flow chart of an exemplary method for crystallization.

FIG. 11 shows a flow chart of an exemplary manufacturing method thatdoes not use crystallization or precipitation. The manufacturing methoduses bacterial fermentation to produce a fermentation culture containingdaptomycin, and then purification of daptomycin using microfiltration,anion exchange chromatography, size exclusion ultrafiltration,hydrophobic interaction chromatography, anion exchange chromatographyfor solvent removal, ultrafiltration for pyrogen removal, reverseosmosis and filling vials with daptomycin. See, e.g., International PCTPublication WO 01/44274, published Jun. 21, 2001, herein incorporated byreference for a detailed description of this type of method.

FIG. 12 shows a flow chart of an exemplary manufacturing method of alipopeptide compound comprising the steps of fermentation,microfiltration, anion exchange chromatography, size exclusionultrafiltration, crystallization or precipitation, crystal orprecipitate drying, and dry filling of vials with the compound. See,e.g., Example 13.

FIG. 13 shows a flow chart of an exemplary manufacturing method of alipopeptide compound comprising the steps of fermentation,microfiltration, anion exchange chromatography, crystallization orprecipitation, crystal or precipitate drying, and dry filling of vialswith the compound. See, e.g., Example 14.

FIG. 14 shows a flow chart of an exemplary manufacturing method of alipopeptide compound comprising the steps of fermentation,microfiltration, size exclusion ultrafiltration, crystallization orprecipitation, crystal or precipitate drying, and dry filling of vialswith the compound. See, e.g., Example 15.

FIG. 15 shows a flow chart of an exemplary manufacturing method of alipopeptide compound comprising the steps of fermentation,microfiltration, crystallization or precipitation, crystal orprecipitate drying, and dry filling of vials with the compound. See,e.g., Example 16.

FIG. 16 depicts the structure of CB-131547, a cyclic lipopeptide analogof daptomycin

DETAILED DESCRIPTION OF THE INVENTION Objects of the Invention

One object of the present invention is to provide methods forcrystallizing or precipitating lipopeptides. In one embodiment, themethods are used to crystallize or precipitate daptomycin or adaptomycin-related lipopeptide. In another embodiment, the methodsincrease the purity of the lipopeptide compared to the purity of thelipopeptide prior to crystallization or precipitation. The methodscomprise the steps of providing an amorphous preparation of alipopeptide and crystallizing or precipitating the lipopeptide underconditions in which the crystalline or precipitated, crystal-likelipopeptide is more pure than the amorphous preparation of thelipopeptide. In one embodiment, the amorphous preparation is no greaterthan 92% pure and the crystalline or crystal-like lipopeptide purifiedtherefrom is at least 95% pure, and may be at least 96%, 97% or 98% ormore pure. In another embodiment, the amorphous preparation is nogreater than 80% pure and the crystalline or crystal-like lipopeptidepurified therefrom is at least 95% pure, and may be at least 96%, 97% or98% or more pure. In another embodiment, the amorphous preparation is nogreater than 60% pure and the crystalline or crystal-like lipopeptidepurified therefrom is at least 95% pure, and may be at least 96%, 97% or98% ore more pure. In yet another embodiment, the amorphous preparationis no greater than 40% pure and the crystalline or crystal-likelipopeptide purified therefrom is at least 95% pure, and may be at least96%, 97% or 98% or more pure. In another embodiment, the amorphouspreparation is no greater than 20% pure and the crystalline orcrystal-like lipopeptide purified therefrom is at least 95% pure, andmay be at least 96%, 97% or 98% or more pure. In a further preferredembodiment, the amorphous preparation is no greater than 10% pure andthe crystalline or crystal-like lipopeptide purified therefrom is atleast 95% pure, and may be at least 96%, 97% or 98% or more pure.

Another object of the invention is to provide processes for making andpurifying a lipopeptide comprising, inter alia, crystallizing orprecipitating the lipopeptides. In one embodiment, the crystallizing orprecipitating steps are used to purify the lipopeptides. In a preferredembodiment, the crystallization or precipitation is performed by batchcrystallized or precipitation. In another embodiment, the process is alarge-scale process for commercial production of a lipopeptide,preferably daptomycin or a daptomycin-related lipopeptide. In oneembodiment, the lipopeptide is produced by fermentation. Thefermentation product is then purified by a variety of purificationtechniques including crystallization or precipitation. In oneembodiment, the crystallization or precipitation step may be used incombination with other purification techniques includingmicrofiltration, size exclusion ultrafiltration and/or anion exchangechromatography. In one embodiment, the crystallization or precipitationstep is used to replace one or more purification techniques that is usedin a purification process that does not use crystallization orprecipitation. In another embodiment, the crystallization orprecipitation step is used to increase purification compared to theother steps without the crystallization or precipitation step. In apreferred embodiment, the method comprises a step of collecting thecrystalline or crystal-like lipopeptide after crystallization orprecipitation.

Another object of the present invention is to provide highly purified,e.g. sterile, crystalline or crystal-like forms of lipopeptides. In oneembodiment, the lipopeptides are daptomycin or a daptomycin-relatedlipopeptide. The crystalline or crystal-like form of the lipopeptide mayhave any crystalline or crystal-like shape including urchin-like(cluster of needles joined together to visually resemble a seaurchin)(see FIG. 2), needle-like (see FIG. 3), rod-like (see FIG. 4),plate-like or flake-like. In one embodiment, the crystalline orcrystal-like lipopeptide has a purity of at least 80%, and may be atleast 85%, 90% pure. In another embodiment, the crystalline orcrystal-like form of the lipopeptide has a purity of at least 95%, andmay be at least 96%, 97%, 98% pure or more.

A further object of the present invention is to provide a pharmaceuticalcomposition comprising a crystalline or crystal-like form of alipopeptide. In one embodiment, the lipopeptide is daptomycin or adaptomycin-related lipopeptide. In one embodiment, the pharmaceuticalcomp. is enterically coated for oral administration or is formulated inthe form of micronized particles or microspheres. In other embodiments,the invention provides methods for administering the pharmaceuticalcompositions to subjects in need thereof.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. The practice of the presentinvention employs, unless otherwise indicated, conventional techniquesof chemistry, biochemistry, biophysics and microbiology and basicterminology used therein.

The term “lipopeptide” refers to a molecule that comprises a lipid-likemoiety covalently linked to a peptide moiety, as well as salts, esters,amides and ethers thereof. The term “lipopeptide” also encompassesprotected forms of lipopeptides in which one or more amino, carboxylateor hydroxyl groups are protected. See, e.g., “Protective Groups inOrganic Synthesis” by Theodora W. Greene, John Wiley and Sons, New York,1981 for examples of protecting groups. In one embodiment, thelipopeptide is an antibiotic. In another embodiment, the lipopeptide isLY 303366, echinocandins, pneumocandins, aculeacins, viscosin,surfactin, plipastatin B1, amphomycin or the lipopeptide derivativedisclosed in U.S. Pat. No. 5,629,288. These lipopeptides are known inthe art. See, e.g., U.S. Pat. No. 5,202,309 and International PCTApplication WO 00/08197. In another embodiment, the lipopeptide is adaptomycin-related molecule. In another embodiment, the lipopeptide isdaptomycin.

A “daptomycin-related molecule” includes, inter alia, daptomycin, A54145or other lipopeptide that is structurally related to daptomycin, such asa daptomycin-related lipopeptide, including all stereoisomers that maybe made at any chiral centers present in these molecules.

A “daptomycin-related lipopeptide” includes, without limitation, alipopeptide disclosed in U.S. Pat. Nos. 4,537,717, 4,482,487, RE32,311,RE32,310, and 5,912,226, currently in reissue as U.S. application Ser.No. 09/547,357. Daptomycin-related lipopeptides also include thosedisclosed in International PCT Publication WO 01/44272, published Jun.21, 2001; International PCT Publication WO 01/44274, published Jun. 21,2001; and International PCT Publication WO 01/44271, published Jun. 21,2001; all of these applications are specifically incorporated herein byreference. The daptomycin-related lipopeptides disclosed in theabove-identified applications relate to synthetic and semisyntheticlipopeptides in which the ornithine and/or kynurine residues, and/or thefatty acid side chain of daptomycin, are modified. Daptomycin-relatedlipopeptides further include an A-21978C₀ antibiotic in which then-decanoyl fatty acid side chain of daptomycin is replaced by an-octanoyl, n-nonanoyl, n-undecanoyl, n-dodecanoyl, n-tridecanoyl orn-tetradecanoyl fatty acid side chain.

The term “daptomycin” refers to the n-decanoyl derivative of the factorA-21978C₀-type antibiotic that contains an α-aspartyl group.“Daptomycin” is synonymous with LY 146032.

The term “anhydro-daptomycin” refers to a daptomycin-related lipopeptidein which an α-aspartyl group of daptomycin is cyclized to a succinimidogroup. See, e.g., the '226 patent for the structure ofanhydro-daptomycin.

The term “β-isomer” or “β-isomer of daptomycin” refers to adaptomycin-related lipopeptide that contains a β-aspartyl group insteadof an α-aspartyl group. See, e.g., the '226 patent for the structure ofβ-isomer of daptomycin.

The term “isolated” refers to a compound or product that is at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the compound present ina mixture. It will be understood that the term “isolated” also refers toa compound that is at least 5-10%, 10-20%, 20-30%, 30-40%, 40-50%,50-60%, 60-70%, 70-80% or 80-90% of the compound present in the mixturegroup. The percentage of compound in a mixture may be measured by anymeans known in the art, as described below for measuring purity of acompound.

“Substantially pure” refers to a sample having at least 95% of a desiredcompound. Preferably, daptomycin is “substantially pure” when at least95% to at least 97% of a sample is daptomycin. Similarly, adaptomycin-related lipopeptide, is “substantially pure” when at least95% to at least 97% of a sample is a daptomycin-related lipopeptide.

Daptomycin or a daptomycin-related lipopeptide is “essentially pure”when at least 98% to at least 99% of a sample is daptomycin or adaptomycin-related lipopeptide, respectively.

Daptomycin or a daptomycin-related lipopeptide is “substantially free”of another compound when the other compound is present in an amount thatis no more than 1% of the amount of the daptomycin or thedaptomycin-related lipopeptide preparation, respectively.

Daptomycin or a daptomycin-related lipopeptide is “essentially free” ofanother compound when the other compound is present in an amount that isno more than 0.5% of the amount of the daptomycin or thedaptomycin-related lipopeptide preparation, respectively.

Daptomycin or a daptomycin-related lipopeptide is “free” of anothercompound when the other compound is present in an amount that is no morethan 0.1% of the amount of the daptomycin or the daptomycin-relatedlipopeptide preparation, respectively. Alternatively, daptomycin or adaptomycin-related lipopeptide is “free” of another compound when thecompound cannot be detected by HPLC under conditions of maximumsensitivity in which a limit of detection is approximately 0.05% or lessof the amount of the daptomycin or the daptomycin-related lipopeptidepreparation, respectively.

“Purified” daptomycin refers to substantially pure daptomycin,essentially pure daptomycin, or a salt thereof, or to daptomycin or asalt thereof which is substantially free, essentially free, or free ofanother compound. Similarly, a “purified” daptomycin-related lipopeptiderefers to a substantially pure daptomycin-related lipopeptide, anessentially pure daptomycin-related lipopeptide, or a salt thereof, orto a daptomycin-related lipopeptide or a salt thereof which issubstantially free, essentially free, or free of another compound.

“Crude” daptomycin refers to daptomycin or a salt thereof that is lessthan 90% pure. Similarly, “crude” daptomycin-related lipopeptide refersto a daptomycin-related lipopeptide or a salt thereof that is less than90% pure.

“Semi-purified” daptomycin refers to daptomycin or a salt thereof thatis at least 90% pure and less than 95% pure. Similarly, “semi-purified”daptomycin-related lipopeptide refers to a daptomycin-relatedlipopeptide or a salt thereof that is at least 90% pure and less than95% pure.

The purity of daptomycin, daptomycin-related lipopeptide or of anotherlipopeptide refers to the lipopeptide prior to its formulation in apharmaceutical composition. The purity of the lipopeptide is referred toby “percent purity.” The measure of purity is not a measure of degree ofcrystallinity of the crystalline preparation. The purity may be measuredby any means including nuclear magnetic resonance (NMR), gaschromatography/mass spectroscopy (GC/MS), liquid chromatography/massspectroscopy (LC/MS) or microbiological assays. One preferred means formeasuring the purity of daptomycin is by analytical high pressure liquidchromatography (HPLC). Two methods of analytical HPLC are described inInternational PCT Publication WO 01/53330, published Jul. 26, 2001,which is herein incorporated specifically by reference.

A “lipopeptide crystal” refers to one or more crystals of a lipopeptideor of a lipopeptide salt. The determination of a lipopeptide as acrystal can be determined by any means including, inter alia, opticalmicroscopy, electron microscopy, x-ray powder diffraction, solid statenuclear magnetic resonance (NMR) or polarizing microscopy. Microscopycan be used to determine the crystal length, diameter, width, size andshape, as well as whether the crystal exists as a single particle or ispolycrystalline.

A lipopeptide or lipopeptide particle is “crystal-like” if it isdetermined to have crystalline characteristics when determined by onemeans, e.g., visually or by optical or polarizing microscopy, but doesnot have crystalline characteristics when determined by another means,e.g., x-ray powder diffraction. A lipopeptide that is “crystal-like” maybe crystalline under certain conditions but may become non-crystallinewhen subjected to other conditions.

A “crystalline lipopeptide” or a “crystalline form of a lipopeptide”refers to a preparation of a lipopeptide or salt thereof that compriseslipopeptide crystals. In one embodiment, a crystalline lipopeptide maycomprise some amount of amorphous lipopeptide. In one embodiment, thecrystalline lipopeptide comprises more than 50% by weight of lipopeptidecrystals. In another embodiment, the crystalline lipopeptide comprisesmore than 60%, 70%, 80%, 90% or 95% of lipopeptide crystals. Thecrystalline lipopeptide may comprise 50-60%, 60-70%, 70-80%, 80-90% or90-95% of lipopeptide crystals. In another embodiment, the crystallinelipopeptide comprises more than 95% of lipopeptide crystals, e.g., atleast 96%, 97%, 98% or 99% lipopeptide crystals or 100% lipopeptidecrystals. The crystalline lipopeptide may also comprise anywhere from95-100% lipopeptide crystals. The percent by weight of lipopeptidecrystals refers to the lipopeptide preparation prior to its formulationin a pharmaceutical composition.

An “amorphous” form of a lipopeptide refers to a lipopeptide preparationthat comprises few or no lipopeptide crystals or crystal-likelipopeptides (or crystal-like particles) as defined herein. In oneembodiment, an amorphous lipopeptide comprises less than 20% by weightof lipopeptide crystals or crystal-like lipopeptides. In anotherembodiment, an amorphous lipopeptide comprises less than 10% by weightof lipopeptide crystals or crystal-like lipopeptides. In anotherembodiment, an amorphous lipopeptide comprises less than 5% by weight oflipopeptide crystals or crystal-like lipopeptides. In a still furtherpreferred embodiment, an amorphous lipopeptide comprises less than 1% byweight of lipopeptide crystals or crystal-like lipopeptides.

“Batch crystallization” refers to a method in which the lipopeptide ofinterest is mixed with the crystallization reagents in solution and thelipopeptide is allowed to crystallize in solution. “Batch precipitation”refers to a method in which the lipopeptide is mixed with precipitationreagents in solution and the lipopeptide is allowed to precipitate insolution. In one embodiment, the crystalline or precipitated preparationis collected from the solution. In another embodiment, the crystallineor precipitated preparation is collected by filtration orcentrifugation.

“Organic precipitant” refers to a polyethylene glycol (PEG) orpolyethylene glycol monomethyl ether (PEG MME) or compounds that arechemically similar.

“Salts” refer to ionic compounds. These ionic compounds may act asprecipitants.

“Low molecular weight alcohols” are organic compounds containing atleast one alcohol functional group, and eight carbon atoms or less. Forexample, low molecular weight alcohols include, without limitation,methanol, isopropanol, and tert-butanol.

“Polyhydric alcohols” refer to compounds that contain more than onealcohol group, and less than eight carbon atoms. Polyhydric alcohols,for example, include, without limitation, 1,6 hexanediol, ethyleneglycol, propylene glycol, glycerol, 1,2-propanediol,2-methyl-2,4-pentanediol and 1,4 butanediol.

“Container” refers to a receptacle for holding goods. For example, acontainer may include, without limitation, an ampule, vial, tube,bottle, or cylinder.

Methods for Producing Purified Lipopeptides

One object of the invention is to provide a method for purifying alipopeptide comprising the steps of providing an amorphous preparationof a lipopeptide and crystallizing or precipitating the lipopeptide. Inone embodiment, the lipopeptide has a higher degree of purity aftercrystallization or precipitation than prior to being subjected tocrystallization or precipitation. Lipopeptides may be crystallized byhanging drop, sitting drop or sandwich drop vapor diffusion,liquid-liquid or free interface diffusion, microdialysis or dialysis,slow solvent evaporation, sublimation, or microbatch or batchcrystallization. In general, a lipopeptide may be precipitated in asimilar way, preferably a lipopeptide is precipitated by batchprecipitation. In a preferred embodiment, the crystallized orprecipitated lipopeptide is daptomycin or a daptomycin-relatedlipopeptide. In a more preferred embodiment, the crystallized orprecipitated lipopeptide is daptomycin.

Lipopeptides may be crystallized or precipitated following the teachingsof this specification. In one embodiment, a lipopeptide can becrystallized or precipitated by providing a solution comprising alipopeptide with a low molecular weight or polyhydric alcohol, a pHbuffering agent and a salt comprising a monovalent or divalent cationand allowing precipitation or crystallization to occur, as discussedfurther infra. In another embodiment, the salt has buffering capacitysuch that an additional pH buffering agent does not have to be presentin the solution. In another embodiment, the salt comprises a divalentcation. In a preferred embodiment, the solution provided does notinclude PEG or PEG-MME or chemically similar compounds. In anembodiment, the method for precipitating or crystallizing thelipopeptide generally comprises the steps of:

a) mixing the lipopeptide with a salt comprising a monovalent ordivalent cation, an optional pH buffering agent and a low molecularweight or polyhydric alcohol; and

b) allowing the lipopeptides to precipitate or crystallize from thesolution under the appropriate temperature conditions.

The samples may be monitored, inter alia, for crystal or precipitateformation by microscopic examination and the yield may be followedspectrophotometrically. In a preferred embodiment, the crystallized orprecipitated lipopeptide is daptomycin or a daptomycin-relatedlipopeptide.

In another embodiment, the lipopeptide can be crystallized by providinga solution comprising a low molecular weight or polyhydric alcohol(s),salts and an organic precipitant as discussed further infra. In a morepreferred embodiment, the crystallized lipopeptide is daptomycin. Ingeneral, for batch crystallization, the lipopeptide is dissolved in asolution and low molecular weight alcohols, salts, buffers and/ororganic precipitants are added to the solution. The samples are thencrystallized under the appropriate temperature conditions, with orwithout stirring. The samples may be monitored, inter alia, for crystalformation by microscopic examination and the yield may be followedspectrophotometrically.

As discussed above, the lipopeptide, preferably daptomycin or adaptomycin-related lipopeptide, is crystallized or precipitated in thepresence of one or more alcohols. In a preferred embodiment, the alcoholis a low molecular weight or polyhydric alcohol. Examples of lowmolecular weight or polyhydric alcohols include, without limitation,methanol, isopropanol, tert-butanol, 1,6 hexanediol, ethylene glycol,propylene glycol, glycerol, 1,2-propanediol, 2-methyl-2,4-pentanedioland 1,4 butanediol. In a preferred embodiment, the alcohol isisopropanol, tert-butanol, glycerol, 1,6-hexanediol, 1,2-propanediol,1,4-butanediol, propylene glycol and/or ethylene glycol. In a morepreferred embodiment, the alcohol is isopropanol.

Salts include, inter alia, magnesium or sodium formate, ammoniumsulfate, ammonium dihydrogen phosphate, calcium acetate, zinc acetate,tri-sodium citrate dihydrate, magnesium acetate, sodium acetate,magnesium chloride, cadmium chloride, ammonium acetate, sodium chlorideand lithium sulfate. In one embodiment, the salt comprises a monovalentcation, e.g., sodium. In a preferred embodiment, the salt comprises adivalent cation. In an even more preferred embodiment, the saltcomprises a calcium cation, a magnesium cation or a manganese cation. Ina further preferred embodiment, the salt comprises a calcium divalentcation. In one embodiment, the salt is calcium chloride, calciumacetate, zinc acetate, sodium citrate, tri-sodium citrate dihydrate,magnesium chloride, lithium sulfate, sodium chloride, magnesium acetate,sodium acetate or a manganese salt, such as manganese acetate ormanganese chloride. In a preferred embodiment, the salt is calciumacetate. Examples of other salts that comprise a divalent cation, suchas a calcium cation, are known in the art, and include, inter alia,those listed in the 2000 Sigma catalog, herein incorporated byreference. Without wishing to be bound to any theory, it is thought thatthe salt cation may neutralize the negative charges on the lipopeptide,e.g., the four carboxylic acids of daptomycin. Organic precipitantsinclude, inter alia, polyethylene glycols (PEGs) that can vary inaverage molecular weight from between 300 and 10,000, or polyethyleneglycol monomethyl ether (PEG-MME). In a preferred embodiment, theorganic precipitant is PEG 300, PEG 600, PEG 2000, PEG 4000, PEG 8000 orPEG 10,000.

The lipopeptide is precipitated or crystallized from a solution that isbuffered to pH 5.0 to 9.5. In one embodiment, prior to being buffered,the solution has a pH of about 1.5, 2.0 or 3.0. In one embodiment,daptomycin or a daptomycin-related lipopeptide is precipitated orcrystallized from a solution of approximately pH 5.5 to approximately pH7.5. In another embodiment, the buffer has a pH of approximately 5.9 toapproximately pH 6.3. In one embodiment, the buffered solution may beobtained by using a pH buffering agent. Examples of pH buffering agentsinclude, without limitation, Tris, phosphate, citrate, HEPES, CHES,sodium acetate or 2-morpholinoethanesulfonic acid (MES), sodium borate,sodium cacodylate, imidazole and tri-sodium citrate dihydrate. In apreferred embodiment, the salt is sodium cacodylate, sodium acetate,tri-sodium citrate dihydrate, HEPES, MES, CHES, imidazole, calciumacetate and Tris-HCl. In a more preferred embodiment, the pH buffer iscalcium acetate pH 6.1, sodium acetate pH 6.1, sodium cacodylate pH 6.5,tri-sodium citrate dihydrate pH 5.6, HEPES pH 7.5, imidazole pH 8, MESpH 6.0, calcium acetate pH 6 and Tris-HCl pH 8.5. In another embodiment,the solution may be buffered by using a salt that also has bufferingcapacity. In a preferred embodiment, the pH buffer is calcium acetate pH6.1.

The lipopeptide is precipitated or crystallized using hanging drop vapordiffusion from a solution containing 2 to 40% low molecular weight orpolyhydric alcohol, 0.001 to 0.5 M salt and 0.005 to 0.2 M pH bufferingagent. In a preferred embodiment, the lipopeptide is precipitated orcrystallized from a solution containing 3 to 30% low molecular weight orpolyhydric alcohol, 0.01 to 0.3 M salt and 0.01 to 0.1 M pH bufferingagent. In a more preferred embodiment, the lipopeptide is precipitatedor crystallized from a solution containing 5 to 20% low molecular weightor polyhydric alcohol, 0.02 to 0.1 M salt and 0.02 to 0.07 M pHbuffering agent. The solution provided may or may not includepolyethylene glycol (PEG) or polyethylene glycol monomethyl ether(PEG-MME).

The lipopeptide is precipitated or crystallized using batchcrystallization from a solution containing 65 to 95% low molecularweight or polyhydric alcohol, 0.001 to 0.5 M salt and 0.001 to 0.2 M pHbuffering agent. In a preferred embodiment, the lipopeptide isprecipitated or crystallized from a solution containing 70 to 90% lowmolecular weight or polyhydric alcohol, 0.005 to 0.04 M salt and 0.005to 0.04 M pH buffering agent. In some embodiments, the lipopeptide iscrystallized from a solution which also comprises 3-8% organicprecipitant. In a more preferred embodiment, the lipopeptide isprecipitated or crystallized from a solution containing 80 to 85% lowmolecular weight or polyhydric alcohol, 0.01 to 0.03 M salt and 0.01 to0.03 M pH buffering agent. In some embodiments, the solution furthercomprises about 4 to 5% organic precipitant, e.g., PEG or PEG-MME. Inother embodiment, the solution provided does not include polyethyleneglycol (PEG) or polyethylene glycol monomethyl ether (PEG-MME).

The lipopeptide is precipitated or crystallized at a temperature fromapproximately 0° C. to approximately 30° C. to obtain precipitate orcrystal formation, respectively. In a preferred embodiment, alipopeptide is crystallized or precipitated at a temperature ofapproximately 20-30° C. In a more preferred embodiment, the mixture iscrystallized or precipitated at approximately 23-28° C. In an even morepreferred embodiment, the mixture is crystallized or precipitated atapproximately 27° C. The mixture may be crystallized or precipitated forany time period that results in crystallization or precipitation,preferably approximately one hour to approximately two weeks. In apreferred embodiment, the mixture is stored for a period ofapproximately three hours to approximately 24 hours, more preferablyapproximately 8-18 hours.

Lipopeptide crystals or crystal-like particles may have a shape that is,without limitation, needle-like, rod-like, urchin-like, flake-like,plate-like or clusters thereof. In one embodiment, lipopeptide crystalsor crystal-like particles are urchin-like, rod-like or needle-like. Theshape of the crystal or crystal-like particle may be determined, interalia, by optical or electron microscopy. In another embodiment,lipopeptide crystals or crystal-like particles may be any size that isat least approximately 0.5 :m in diameter in any one dimension. In amore preferred embodiment, lipopeptide crystals or crystal-like particleare at least 5 μm, more preferably at least 10 μm. In an even morepreferred embodiment, the lipopeptide crystals or crystal-like particlesare at least 50 μm, more preferably at least 100 μm. The size of thecrystal may be determined by any method known to one having ordinaryskill in the art. See, e.g., United States Pharmacopeia (USP), pp.1965-67.

The properties of a crystalline or crystal-like lipopeptide may bedetermined by any method known to one having ordinary skill in the art.The properties that can be determined include the crystalline orcrystal-like lipopeptide's size, shape, birefringence properties, powderx-ray diffraction properties, solid state NMR properties, meltingtemperature and stability to heat, light, humidity, and degradation. Ina preferred embodiment, one having ordinary skill in the art maydetermine whether a lipopeptide is crystalline by powder x-raydiffraction. Powder x-ray diffraction is highly useful for determiningwhether a preparation is crystalline when the sample is arandomly-oriented collection of small crystals. Diffraction by a mass ofrandomly-oriented microcrystals produces a series of lines or rings(dependent of the detector) characteristic of the molecule studied andits structure. In a preferred embodiment, powder diffraction is measuredby an Automated Powder Diffraction instrument in order to determinewhether a lipopeptide is crystalline. See, e.g., Atkins et al., PhysicalChemistry, pp. 710-716 (1978), herein incorporated by reference for adiscussion of the Debye-Scherrer method for powder diffraction. Anypowder diffractometer instrument known in the art that is equipped withany detector for powder diffraction that known in the art could be usedto measure the diffraction pattern.

In a preferred embodiment of the invention, a lipopeptide iscrystallized or precipitated using a buffering agent betweenapproximately pH 5.0 and 9.5, a salt and an alcohol at a temperature ofapproximately 24-28° C. for a period of approximately three to 24 hours.In a preferred embodiment, the salt is a buffering agent and comprises adivalent cation and the alcohol is a low molecular weight alcohol, andthe pH is between approximately pH 5.5 and 7.5. In an even morepreferred embodiment, the salt is a calcium salt, the alcohol isisopropanol and the pH is between approximately pH 5.9 and 6.3. Inembodiments were the solution includes an organic precipitant,preferably the organic precipitant is PEG 4000 or PEG 8000. In anotherembodiment the lipopeptide is precipitated or crystallized from asolution containing 12 to 18% glycerol, 0.3 to 0.8 m salt, 0.03 to 0.08m pH buffering agent, and 12-18% PEG 600. In a still further preferredembodiment, the lipopeptide is daptomycin or a daptomycin-relatedlipopeptide. Examples 2-3 provide methods for precipitating a highlypure crystal-like daptomycin. One having ordinary skill in the art,following the teachings of the instant specification, may modify thecrystallization/precipitation conditions provided in the examples tocrystallize or precipitate daptomycin, daptomycin-related lipopeptides,or other lipopeptides of interest. Further, although the teachings ofthe instant specification describe the use of a single crystallizationor precipitation step in a process for purifying a lipopeptide, onehaving ordinary skill in the art following the teachings of thespecification may use multiple crystallization or precipitation steps ina process for purifying a lipopeptide. It may be advantageous to employmultiple rounds of crystallization or precipitation as disclosed hereinin order to further increase purity of the lipopeptide.

After crystallization or precipitation, one may collect the crystallinematerial or crystal-like precipitate by any method known in the art. Ina preferred embodiment, the crystalline material or crystal-likeprecipitate is collected by centrifugation or filtration. In an evenmore preferred embodiment, the crystalline material or crystal-likeprecipitate is collected by filtration because filtration is easilyincorporated into a large-scale process for producing a lipopeptide.After the crystalline material or crystal-like precipitate is collected,it may be washed to remove excess crystallizing or precipitatingreagents. Any wash solvent known in the art may be chosen so long as itdoes not appreciably dissolve the crystalline material or crystal-likeprecipitate. An example of a wash solvent is provided in Example 12.After the crystalline material or crystal-like precipitate is washed, itmay be dried by any method known in the art. Examples of drying methodsinclude air-drying, lyophilization (freeze-drying) or desiccation. In apreferred method, the crystalline material or crystal-like precipitateis desiccated. See, e.g., Example 12. In another embodiment, thecrystalline lipopeptide's stability may be determined by its residualantibiotic activity or its degradation. The antibiotic activity may bemeasured in a standard agar-diffusion assay against various bacterialstrains. See, e.g., Example 32 of U.S. Pat. No. 4,537,717, specificallyincorporated herein by reference. The amount of degradation can bemeasured by, inter alia, HPLC analysis, such as that described inInternational PCT Publication WO 01/53330, published Jul. 26, 2001. In apreferred embodiment, the stability of the crystalline lipopeptide isgreater than that of the amorphous form of the lipopeptide. Thestability of the crystalline lipopeptide may be determined by exposingthe crystalline lipopeptide and an amorphous form thereof to heat,light, humidity, and measuring the degree of degradation of thecrystalline form to that of the amorphous form.

Degradation of the lipopeptide may be measured by determining thebiological activity of the lipopeptide or any applicable physicalparameter. In one embodiment, degradation may be measured by determininga particular biological activity of a lipopeptide after it has beensubjected to heat, light, humidity, changes in pH or extreme pH, andcomparing it to the same biological activity of the lipopeptide prior toany tests of stability. The amount of degradation may be determined, forexample, by determining the percentage of biological activity remainingafter the test of stability. The percentage of remaining biologicalactivity may be compared to that of an amorphous form of the lipopeptidethat has been subjected to the same test. In one embodiment, if thelipopeptide is an antibiotic, the crystalline lipopeptide may be testedfor its antibiotic activity both prior to and after a test of itsstability and compared to an amorphous form that has been tested priorto and after a degradation test. In a preferred embodiment, thelipopeptide is daptomycin or a daptomycin-related lipopeptide, and thebiological activity test determines the amount of antibiotic activity ofthe lipopeptides against gram-positive bacteria.

Degradation of a lipopeptide may also be measured by a physical assay.In one embodiment, degradation may be measured by determining thepercentage of intact crystalline lipopeptide that remains after a testof its stability. The percentage of remaining intact lipopeptide may becompared to that of an amorphous form of the lipopeptide that has beensubjected to the same test for stability. In a preferred embodiment, thedegradation of the lipopeptide may be measured by HPLC, ultravioletspectroscopy, infrared spectroscopy, NMR, or mass spectroscopy. In aneven more preferred embodiment, HPLC is used to determine the percentageof intact lipopeptide that remains after a crystalline form of alipopeptide has been subjected to a test of its stability.

Without wishing to be bound by any theory, applicants believe thatdaptomycin is crystallized by the methods described above. However, itis thought that washing and/or drying the daptomycin crystals causes thedaptomycin crystalline material to revert to a non-crystalline but stillcrystal-like form. Nevertheless, even if the methods described aboveonly precipitate rather than crystallize the daptomycin or otherlipopeptide, the methods still are advantageous because the methodspurify the lipopeptide.

The invention also provides a crystalline or crystal-like lipopeptideproduced by the above-described methods. In one embodiment, thecrystalline or crystal-like lipopeptide comprises a lower amount of oneor more impurities compared to the lipopeptide before crystallization orprecipitation. In one embodiment, crystalline or crystal-likelipopeptide is daptomycin that comprises a lower level ofanhydro-daptomycin and/or the β-isomer of daptomycin compared todaptomycin before crystallization or precipitation. In anotherembodiment, crystalline or crystal-like daptomycin comprises a lowerlevel of all impurities compared to amorphous daptomycin. Similarly, inanother embodiment, the crystalline or crystal-like lipopeptide is adaptomycin-related lipopeptide, as described above, which comprises alower level of one or more impurities compared to an amorphous form ofthe daptomycin-related lipopeptide. In yet another embodiment, thecrystalline or crystal-like daptomycin-related lipopeptide comprises alower level of all impurities compared to an amorphous form of thedaptomycin-related lipopeptide.

The crystalline or crystal-like lipopeptide produced by the methoddescribed above likely comprises monovalent or divalent cations andwater. In a preferred embodiment, the crystalline or crystal-likelipopeptide is daptomycin or daptomycin-related lipopeptide thatcomprises a divalent cation. In a more preferred embodiment, thedivalent cation is a calcium cation. In an even more preferredembodiment, the crystalline or crystal-like daptomycin ordaptomycin-related lipopeptide comprises approximately 1-10% by weightof a divalent calcium cation and approximately 0-15% by weight of wateras determined by atomic absorption or thermal gravity analysis. In afurther preferred embodiment, the crystalline or crystal-likelipopeptide is daptomycin that comprises approximately 5% by weight of adivalent calcium cation and approximately 10% by weight of water; byHPLC analysis, the purity of the crystalline or crystal-like daptomycinis at least 95%, 96%, 97% or 98% or is any purity between 95-98%,relative to related substances and organic contaminants. Alternatively,the crystalline or crystal-like daptomycin or daptomycin-relatedlipopeptide comprises a monovalent cation such as sodium. Withoutwishing to be bound by any theory, it is thought that daptomycin or adaptomycin-related lipopeptide may form a salt with the monovalent ordivalent cation when it crystallizes or precipitates.

The crystalline form of the lipopeptide may exhibit an increasedsolubility in a solution or an increased rate of reconstitution in asolution than an amorphous form of the lipopeptide. One may measurewhether the crystalline lipopeptide exhibits an increased solubility orincreased reconstitution rate by any method known in the art. Forinstance, one may dissolve a defined amount of a crystalline lipopeptidein an aqueous solution and measure the concentration of the dissolvedlipopeptide and compare it to the concentration of dissolved lipopeptidethat has been prepared by dissolving the same amount of amorphouslipopeptide in an aqueous solution. Similarly, one may measure thereconstitution rate of a crystalline lipopeptide by adding thecrystalline lipopeptide to an aqueous solution and then measuring theconcentration of dissolved lipopeptide over time and comparing it to thereconstitution rate of an amorphous lipopeptide that has been measuredin the same way. The concentration of lipopeptide is measured by HPLC.

The methods described above provide for the production of crystalline orcrystal-like lipopeptides that are more pure than the amorphouslipopeptide from which they are crystallized or precipitated. In oneembodiment, the lipopeptide is daptomycin or a daptomycin-relatedlipopeptide. In another embodiment, daptomycin or a daptomycin-relatedlipopeptide has a purity of no more than 92% before crystallization andhas a purity of at least approximately 95%, 96%, 97% or 98% purity, orany purity between 95-98%, after crystallization or precipitation as acrystal-like lipopeptide. In a still further preferred embodiment,daptomycin or a daptomycin-related lipopeptide has a purity of no morethan 90% before crystallization and has a purity of approximately atleast 97% or 98% after crystallization.

In another embodiment, the daptomycin has a purity of no more than 80%,preferably no more than 70% and more preferably no more than 60% puritybefore crystallization or precipitation, and has at least approximately95%, 96%, 97% or 98% purity, or any purity between 95-98%, afterpurification. In another embodiment, the daptomycin has a purity of nomore than 50%, preferably no more than 40%, more preferably no more than30% purity before crystallization and has at least approximately 95%,96%, 97% or 98% purity, or any purity between 95-98%, after purificationby crystallization or precipitation. Further preferred is an embodimentin which daptomycin has a purity of no more than 20%, more preferably nomore than 15%, even more preferably no more than 10% purity beforecrystallization and has at least approximately 95%, 96%, 97% or 98%purity, or any purity between 95-98%, after purification.

In a more preferred embodiment, the lipopeptide is daptomycin. Adaptomycin preparation may be obtained by any method disclosed, e.g., inany one U.S. Pat. Nos. RE32,333, RE32,455, 4,800,157, RE32,310,RE32,311, 4,537,717, 4,482,487, 4,524,135, 4,874,843, 4,885,243 or U.S.Pat. No. 5,912,226, which are herein incorporated specifically byreference. A daptomycin preparation may also be obtained by one of themethods described in International PCT Publication WO 01/53330,published Jul. 26, 2001. After the lipopeptide preparation is prepared,the lipopeptide preparation is crystallized or precipitated followingthe teachings of the specification described herein to produce acrystalline or crystal-like lipopeptide that is more pure or thatcontains lower levels of specific impurities, e.g., anhydro-daptomycin,than the lipopeptide preparation from which it is prepared.

Processes for Producing Purified Lipopeptides from Fermentation Cultures

Another embodiment of the present invention is drawn to a processcombining process chromatography steps and crystallization orprecipitation to produce a purified lipopeptide. In a preferredembodiment, the method comprises the steps of producing a lipopeptide byany method known in the art, such as fermentation of anaturally-occurring or recombinant organism, and then subjecting thelipopeptide preparation to any one or more purification methods such asmicrofiltration, anion exchange chromatography, hydrophobic interactionchromatography, and/or size exclusion chromatography (either viatraditional size exclusion chromatographic media or via ultrafiltration)to produce a lipopeptide preparation that has been partially purified,and then crystallizing or precipitating the lipopeptide preparation toobtain a purified crystalline or crystal-like lipopeptide. In apreferred embodiment, the lipopeptide is daptomycin or adaptomycin-related lipopeptide. The steps regarding fermentation,microfiltration, anion exchange chromatography, hydrophobic interactionchromatography and ultrafiltration are disclosed in the art, e.g., inany one U.S. Pat. Nos. RE32,333, RE32,455, 4,800,157, RE32,310,RE32,311, 4,537,717, 4,482,487, 4,524,135, 4,874,843, 4,885,243 or U.S.Pat. No. 5,912,226, in International Publication WO 01/53330, publishedJul. 26, 2001.

The method optionally comprises the step of collecting and/or washingthe crystalline or crystal-like material after the crystallization orprecipitation step. In a preferred embodiment, the crystallinelipopeptide preparation may be collected by filtration. In anotherembodiment, the crystalline or crystal-like material is dried.

In one embodiment, the purification method comprises fermentingStreptomyces roseosporus to obtain a fermentation culture containingdaptomycin. In one embodiment, the S. roseosporus may be fermented asdescribed in U.S. Pat. No. 4,885,243. In another embodiment, thefermentation conditions in which the A-21978C₀-containing crude productis produced by Streptomyces roseosporus is altered in order to increasedaptomycin production and decrease impurities and related contaminantsproduced by the S. roseosporus fermentation culture as described inInternational PCT Publication WO 01/53330, published Jul. 26, 2001. TheWO 01/53330 publication describes fermenting S. roseosporus as describedin the '243 patent with the modification that the decanoic acid feed iskept at the lowest levels possible without diminishing the overall yieldof the fermentation.

Alternatively, daptomycin may be obtained by fermenting a bacterialstrain or other producing organism that recombinantly producesdaptomycin. In one embodiment, the recombinant bacterial strain or otherrecombinant organism comprises the daptomycin biosynthetic gene cluster.In another embodiment, the daptomycin biosynthetic gene cluster or aportion thereof is introduced into the organism or bacterial strain viaa bacterial artificial chromosome (BAC). In another embodiment, therecombinant bacterial strain used is S. roseosporus or S. lividanscomprising a BAC containing the daptomycin biosynthetic gene cluster.U.S. Provisional Application 60/272,207, filed Feb. 28, 2001 describesthe daptomycin biosynthetic gene cluster from S. roseosporus and usesthereof, and is hereby incorporated by reference in its entirety.

After fermentation, the fermentation broth is clarified bycentrifugation, microfiltration or extraction, as is known in the art oras described in the WO 01/53330 publication. In a preferred embodiment,the clarification is performed by microfiltration. See, e.g., Examples13-16 and FIGS. 11-15. FIG. 11 shows an exemplary manufacturing processthat does not use crystallization or precipitation.

After the fermentation broth is clarified, the concentration ofdaptomycin in the broth is approximately 5-10%. In one embodiment of theinvention, the daptomycin preparation is subjected to acrystallization/precipitation method described above directly subsequentto microfiltration. In one embodiment, crystallization or precipitationis performed under sterile conditions. After crystallization orprecipitation is complete, the crystalline or crystal-like daptomycin isoptionally collected, washed and dried, as described in further detailbelow. The dry bulk active drug may then be used to dry fill sterilevials. See, e.g., Example 16 and FIG. 12.

After clarification of the fermentation broth, the lipopeptide may beenriched in the preparation by anion exchange chromatography, as isknown in the art or as described in the WO 01/53330 publication orherein. See, e.g., Examples 13-14 and FIGS. 12-13. After anion exchangechromatography, the purity of daptomycin in the broth is approximately35-40%. In one embodiment of the invention, the daptomycin preparationis then subjected to a crystallization or precipitation method describedabove directly subsequent to anion exchange chromatography. In oneembodiment, crystallization or precipitation is performed under sterileconditions. After crystallization or precipitation is complete, thecrystalline or crystal-like daptomycin is optionally collected, washedand dried as described below. The dry bulk active drug may then be usedto dry fill sterile vials. See, e.g., Example 14 and FIG. 13.

In another embodiment of the invention, the daptomycin preparation issubjected to size exclusion ultrafiltration after anion exchangechromatography. Size exclusion ultrafiltration is described in the WO01/53330 publication. The application published Jul. 26, 2001 describesa method of depyrogenating, filtering and concentrating the daptomycinusing an ultrafiltration membrane of 10,000 to 30,000 nominal molecularweight (NMW). The application discloses a method in which thelipopeptide passes through the ultrafiltration membrane while largemolecular weight impurities, such as endotoxins, are retained by thefilter. After the lipopeptide has passed through the membrane, the pH,temperature and/or salt concentration of the lipopeptide solution arealtered such that the lipopeptides form micelles. The lipopeptidesolution is then filtered on the ultrafiltration membrane underconditions in which the lipopeptide micelles are retained on themembrane while smaller impurities pass through the filter. In thismanner, the lipopeptide is further purified. The application disclosesthe conditions under which lipopeptide micelles may be formed anddisassociated as well as methods for filtering the lipopeptide solutionto obtain a more purified lipopeptide application. In an even morepreferred embodiment, the lipopeptide is daptomycin or adaptomycin-related lipopeptide. The lipopeptide may then becrystallized, as described herein. After both anion exchangechromatography and size exclusion ultrafiltration, daptomycin purity isapproximately 80-90%. As discussed above, the daptomycin preparation isthen subjected to a crystallization/precipitation method describedabove, preferably under sterile conditions. The crystalline orcrystal-like daptomycin may be optionally collected, washed, dried andused to dry fill vials as described below. See, e.g., Example 13 andFIG. 12.

In another embodiment of the invention, the crude daptomycin preparationis subjected to size exclusion ultrafiltration without anion exchangechromatography. After size exclusion ultrafiltration, daptomycin purityis approximately 35-40%. The lipopeptide may then be crystallized orprecipitated as described herein, preferably by sterile methods. Asdiscussed above, the crystalline or crystal-like daptomycin may becollected, washed, dried and used to dry fill sterile vials. See, e.g.,Example 15 and FIG. 14.

In an alternative embodiment, the lipopeptide preparation is subjectedto hydrophobic interaction chromatography (HIC), such as is described inthe WO 01/53330 publication, after either the anion exchangechromatography or the size exclusion filtration. The lipopeptide maythen be crystallized or precipitated as described herein.

After crystallization or precipitation, the crystalline or crystal-likelipopeptide may be collected by a method described herein, e.g., byfiltration or centrifugation. The crystalline or crystal-likelipopeptide is optionally washed to remove residual crystallization orprecipitation solvent. A method of washing crystals or crystal-likematerial are described below. See, e.g., Example 3. The washed orunwashed crystal or crystal-like material may be dried. The drying maybe performed by any method known in the art, including, withoutlimitation, vacuum drying, spray drying, tray drying or lyophilization.In one embodiment, the drying is performed under sterile conditions. Inanother embodiment, the drying is performed by vacuum drying. In a morepreferred embodiment, the drying is performed using a 0.65 m³ KleinHastelloy-B double cone vacuum dryer or an equivalent apparatus.

The dried crystalline or crystal-like lipopeptide is stable and iseasily stored.

In one embodiment, vials are filled with any convenient amount of thedried crystalline or crystal-like lipopeptide. In one embodiment, thevials are filled under sterile conditions and then stoppered. In anotherembodiment, the vials are filled with 50 to 5000 mg each of the driedcrystalline or crystal-like lipopeptide. In another embodiment, thevials are filled with 100 to 1000 mg each. In another embodiment, thevials are filled with 200 to 500 mg each. In another embodiment, thedried crystalline or crystal-like lipopeptide is used for bulk packagingof the lipopeptide. The bulk packaging is usually greater than 5000 mgeach of the dried crystalline or crystal-like lipopeptide. In oneembodiment, the bulk packaging is performed under sterile conditions.

In one embodiment, the crystallization or precipitation step isperformed under sterile conditions. In this embodiment, sterilecrystallization or precipitation reagents and a sterile, controlledworking environment are used. In one embodiment, the lipopeptide isfiltered on a ultrafiltration membrane, as disclosed above, before beingmixed with the sterile crystallization/precipitation reagents. Aftercrystallization or precipitation, the crystalline or crystal-likelipopeptide preparation is collected by centrifugation or filtrationunder sterile conditions. In one embodiment, the lipopeptide preparationis collected by sterile filtration. In another embodiment, thecrystalline or crystal-like lipopeptide is sterilized after it has beencollected. Methods of sterile crystallization, precipitation andfiltration as well as methods of sterilizing a final pharmaceuticalproduct are known in the art. See, e.g., Remington: The Science andPractice of Pharmacy, Easton, Pa.: Mack Publishing Company (1995), pp.1474-1487, herein incorporated by reference.

In another embodiment, the crystalline or crystal-like lipopeptide isnot dried. In this embodiment, the crystalline or crystal-likelipopeptide is preferably stored in a solution that preserves thecrystalline or crystal-like nature of the lipopeptide. Vials may befilled with the lipopeptide and solution under sterile or nonsterileconditions. In one embodiment, the conditions are sterile.Alternatively, the crystalline or crystal-like lipopeptide and solutionmay be used to fill bulk packaging.

FIGS. 10 and 11 provide flowcharts describing an exemplary daptomycinmanufacturing protocol using crystallization. The incorporation ofsterile crystallization into the manufacturing protocol shortens theprotocol considerably and eliminates 3 to 4 steps in the process.

Crystalline or Crystal-Like Lipopeptides, Pharmaceutical Compositionsand Methods of Use Thereof

Another object of the instant invention is to provide crystalline orcrystal-like lipopeptides or salts thereof, as well as pharmaceuticalformulations comprising a crystalline or crystal-like lipopeptide or itssalts. In one embodiment, the crystalline or crystal-like lipopeptide isdaptomycin. However, all reference herein to crystalline or crystal-likelipopeptides specifically contemplates daptomycin, a daptomycin-relatedmolecule, including, inter alia, daptomycin, A54145 and adaptomycin-related lipopeptide, as disclosed above.

Daptomycin crystals or crystal-like particles, as well as otherlipopeptide crystals or crystal-like particles may have a shape such as,inter alia, a needle-like shape, a plate-like shape, a lath-like shape,an equant-like shape, an urchin-like shape or a rod-like shape. In oneembodiment, daptomycin crystals or crystal-like particles have anurchin-like, needle-like or rod-like shape. The size of the crystals orcrystal-like particles may range from approximately 0.5 μm to greaterthan 100 μm. In one embodiment, the particle size is at least 5 μm orgreater. In a more preferred embodiment, the particle size is at least10 μm or greater, more preferably at least 50 μm. In an even morepreferred embodiment, the particle size is at least 100 μm.

Further, in one embodiment, daptomycin crystals have an x-raydiffraction pattern as shown in FIGS. 6, 7 and 8. In another embodiment,the lipopeptide crystal exhibits a different melting point than theamorphous form of the lipopeptide.

In one embodiment of the invention, a crystalline form of a lipopeptideexhibits a stability that is equal to or greater than the amorphous formof the lipopeptide. In a preferred embodiment, the crystalline form isdaptomycin or a daptomycin-related lipopeptide. In another preferredembodiment, the crystalline lipopeptide is sterile. In another preferredembodiment, the stability of the crystalline lipopeptide is greater thanthe amorphous form of the lipopeptide. The crystalline lipopeptide mayexhibit higher stability to heat, light, degradation or humidity thanthe amorphous form. The stability of the lipopeptide may be measured byany means including, e.g., antibiotic activity, degradation of thelipopeptide or conversion of daptomycin to anhydro-daptomycin or theβ-isomer of daptomycin. In another embodiment of the invention, thecrystalline form of the lipopeptide may be more quickly reconstituted inaqueous solution than the amorphous form of the lipopeptide.

Crystalline or crystal-like lipopeptides, such as daptomycin or adaptomycin-related lipopeptide, pharmaceutically-acceptable salts,esters, amides, ethers and protected forms thereof, can be formulatedfor oral, intravenous, intramuscular, subcutaneous, aerosol, topical orparenteral administration for the therapeutic, empirical or prophylactictreatment of diseases, particularly bacterial infections. Referenceherein to “crystalline or crystal-like lipopeptides” or “crystalline orcrystal-like daptomycin” includes pharmaceutically acceptable saltsthereof. Crystalline or crystal-like lipopeptides, such as daptomycin,may be particularly advantageous for pharmaceutical compositions becausethey can be easily formulated as micronized particles of microspheres,which permits the facile preparation of enterically coated lipopeptidesfor oral delivery, pharmaceutical compositions for aerosol delivery to,e.g., the lung, and the preparation of lipopeptides formulations forsustained release. Crystalline or crystal-like lipopeptides andcrystalline or crystal-like daptomycin may also be more readilydissolved in aqueous solution.

Crystalline or crystal-like lipopeptides, including daptomycin ordaptomycin-related lipopeptides can be formulated using anypharmaceutically acceptable carrier or excipient that is compatible withdaptomycin or with the lipopeptide of interest. See, e.g., Handbook ofPharmaceutical Additives: An International Guide to More than 6000Products by Trade

Name, Chemical, Function, and Manufacturer, Ashgate Publishing Co.,eds., M. Ash and I. Ash, 1996; The Merck Index: An Encyclopedia ofChemicals, Drugs and Biologicals, ed. S. Budavari, annual; Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.;Martindale: The Complete Drug Reference, ed. K. Parfitt, 1999; andGoodman & Gilman's The Pharmaceutical Basis of Therapeutics, PergamonPress, New York, N.Y., ed. L. S. Goodman et al.; the contents of whichare incorporated herein by reference, for a general description of themethods for administering various antimicrobial agents for humantherapy. Compounds of this invention can be mixed with conventionalpharmaceutical carriers and excipients and used in the form of tablets,capsules, elixirs, suspensions, syrups, wafers, creams and the like.Compounds of this invention may also be mixed with other therapeuticagents and antibiotics, such as discussed herein. The compositionscomprising a compound of this invention will contain from about 0.1 toabout 90% by weight of the active compound, and more generally fromabout 10 to about 30%.

The compositions of the invention can be delivered using controlled(e.g., capsules) or sustained release delivery systems (e.g.,bioerodable matrices). Exemplary delayed release delivery systems fordrug delivery that are suitable for administration of the compositionsof the invention are described in U.S. Pat. No. 4,452,775 (issued toKent), U.S. Pat. No. 5,239,660 (issued to Leonard), U.S. Pat. No.3,854,480 (issued to Zaffaroni).

The compositions may contain common carriers and excipients, such ascorn starch or gelatin, lactose, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.The compositions may contain croscarmellose sodium, microcrystallinecellulose, corn starch, sodium starch glycolate and alginic acid.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallicstearates, stearic acid, silicone fluid, talc, waxes, oils and colloidalsilica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. It may also be desirable to adda coloring agent to make the dosage form more aesthetic in appearance orto help identify the product.

For oral use, solid formulations such as tablets and capsules areparticularly useful. Sustained release or enterically coatedpreparations may also be devised. In another embodiment, crystalline orcrystal-like lipopeptides may be supplied in combination with a carriercomposition that enhances the oral availability of the lipopeptide. In apreferred embodiment, the crystalline or crystal-like lipopeptide isdaptomycin. For pediatric and geriatric applications, suspensions,syrups and chewable tablets are especially suitable. For oraladministration, the pharmaceutical compositions are in the form of, forexample, a tablet, capsule, suspension or liquid. The pharmaceuticalcomposition is preferably made in the form of a dosage unit containing atherapeutically-effective amount of the active ingredient. Examples ofsuch dosage units are tablets and capsules. For therapeutic purposes,the tablets and capsules which can contain, in addition to the activeingredient, conventional carriers such as binding agents, for example,acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth;fillers, for example, calcium phosphate, glycine, lactose, maize-starch,sorbitol, or sucrose; lubricants, for example, magnesium stearate,polyethylene glycol, silica, or talc; disintegrants, for example, potatostarch, flavoring or coloring agents, or acceptable wetting agents. Oralliquid preparations generally are in the form of aqueous or oilysolutions, suspensions, emulsions, syrups or elixirs may containconventional additives such as suspending agents, emulsifying agents,non-aqueous agents, preservatives, coloring agents and flavoring agents.Oral liquid preparations may comprise lipopeptide micelles or monomericforms of the lipopeptide. Examples of additives for liquid preparationsinclude acacia, almond oil, ethyl alcohol, fractionated coconut oil,gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin,methyl cellulose, methyl or propyl para-hydroxybenzoate, propyleneglycol, sorbitol, or sorbic acid.

For intravenous (IV) use, a water soluble form of a compound of thisinvention can be dissolved in any of the commonly used intravenousfluids and administered by infusion. Intravenous formulations mayinclude carriers, excipients or stabilizers including, withoutlimitation, calcium, human serum albumin, citrate, acetate, calciumchloride, carbonate, and other salts. Intravenous fluids include,without limitation, physiological saline or Ringer's solution.Daptomycin or other lipopeptides also may be placed in injectors,cannulae, catheters and lines.

Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The crystalline or crystal-likelipopeptides can be dissolved in polyethylene glycol, propylene glycol,ethanol, corn oil, benzyl alcohol, sodium chloride, and/or variousbuffers. For intramuscular, parenteral or intravenous preparations, asterile formulation of a crystalline or crystal-like lipopeptidecompound or a suitable soluble salt form of the compound, for examplethe hydrochloride salt, can be dissolved and administered in apharmaceutical diluent such as Water-for-Injection (WFI), physiologicalsaline or 5% glucose. A suitable insoluble form of the crystalline orcrystal-like lipopeptide also may be prepared and administered as asuspension in an aqueous base or a pharmaceutically acceptable oil base,e.g., an ester of a long chain fatty acid such as ethyl oleate.

Injectable depot forms may be made by forming microencapsulated matricesof the crystalline or crystal-like lipopeptide in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in microemulsionsthat are compatible with body tissues.

For topical use the compounds of the present invention can also beprepared in suitable forms to be applied to the skin, or mucus membranesof the nose and throat, and can take the form of creams, ointments,liquid sprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient. For topical preparations, a sterile formulation comprising acrystalline or crystal-like lipopeptide, such as crystalline orcrystal-like daptomycin, a suitable salt form thereof, may beadministered in a cream, ointment, spray or other topical dressing.Topical preparations may also be in the form of bandages that have beenimpregnated with a lipopeptide composition.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

For aerosol preparations, a sterile formulation of a crystalline orcrystal-like lipopeptide or a salt form of the compound may be used ininhalers, such as metered dose inhalers, and nebulizers. Aerosolizedforms may be especially useful for treating respiratory infections, suchas pneumonia and sinus-based infections.

Alternatively, the compounds of the present invention can be in powdercrystalline or crystal-like form for reconstitution in the appropriatepharmaceutically acceptable carrier at the time of delivery. In anotherembodiment, the unit dosage form of the compound can be a solution ofthe compound or a salt thereof in a suitable diluent in sterile,hermetically sealed ampules. The concentration of the compound in theunit dosage may vary, e.g. from about 1 percent to about 50 percent,depending on the compound used and its solubility and the dose desiredby the physician. If the compositions contain dosage units, each dosageunit preferably contains approximately from 10-5000 mg of the activematerial, more preferably 50 to 1000 mg, and even more preferably 100 to500 mg. For adult human treatment, the dosage employed preferably rangesfrom 100 mg to 3 g, per day, depending on the route and frequency ofadministration.

In a further aspect, this invention provides a method for treating aninfection caused by a gram-positive bacteria in a subject. In apreferred embodiment, the method may be used to treat an infectioncaused by a gram-positive bacteria. The term “treating” is defined asadministering, to a subject, a therapeutically-effective amount of acompound of the invention, both to prevent the occurrence of aninfection and to control or eliminate an infection, e.g., an establishedinfection. The term “subject”, as described herein, is defined as amammal, a plant or a cell culture. As used herein, the phrase“therapeutically-effective amount” means an amount of daptomycin,daptomycin-related lipopeptide or other antibacterial lipopeptideaccording to the present invention that prevents the onset, alleviatesthe symptoms, or stops the progression of a bacterial infection. In apreferred embodiment, a subject is a human or other animal patient inneed of lipopeptide treatment. An established infection may be one thatis acute or chronic. An effective dose is generally between about 0.1and about 75 mg/kg crystalline or crystal-like lipopeptide, such ascrystalline or crystal-like daptomycin or daptomycin-relatedlipopeptide, or a pharmaceutically acceptable salt thereof. A preferreddose is from about 1 to about 25 mg/kg of crystalline or crystal-likedaptomycin or daptomycin-related lipopeptide or a pharmaceuticallyacceptable salt thereof. A more preferred dose is from about 1 to 12mg/kg crystalline or crystal-like daptomycin, a crystalline orcrystal-like daptomycin-related lipopeptide or a pharmaceuticallyacceptable salt thereof. An even more preferred dose is about 3 to 8mg/kg crystalline or crystal-like daptomycin or daptomycin-relatedlipopeptide or a pharmaceutically acceptable salt thereof. Exemplaryprocedures for delivering an antibacterial agent are described in U.S.Pat. No. 5,041,567, issued to Rogers and in International PCTPublication WO 95/05384, the entire contents of which documents areincorporated in their entirety herein by reference.

The crystalline or crystal-like lipopeptide, e.g., daptomycin, can beadministered as a single daily dose or in multiple doses per day. Thetreatment regime may require administration over extended periods oftime, e.g., for several days or for from two to four weeks. The amountper administered dose or the total amount administered will depend onsuch factors as the nature and severity of the infection, the age andgeneral health of the patient, the tolerance of the patient to thelipopeptide and the microorganism or microorganisms involved in theinfection. A method of administration is disclosed in WO 00/18419,published Apr. 6, 2000, herein incorporated by reference.

The methods of the present invention comprise administering a compoundof the invention, or a pharmaceutical composition thereof to a patientin need thereof in an amount that is efficacious in reducing oreliminating the gram-positive bacterial infection. The lipopeptide maybe administered orally, parenterally, by inhalation, topically,rectally, nasally, buccally, vaginally, or by an implanted reservoir,external pump or catheter. The lipopeptide may be prepared for opthalmicor aerosolized uses. Compounds of the invention, or pharmaceuticalcompositions thereof also may be directly injected or administered intoan abscess, ventricle or joint. Parenteral administration includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, cisternal, intrathecal, intrahepatic, intralesional andintracranial injection or infusion. In a preferred embodiment,crystalline or crystal-like daptomycin, daptomycin-related lipopeptideor other lipopeptide is administered intravenously, subcutaneously ororally.

The method of the instant invention may be used to treat a patienthaving a bacterial infection in which the infection is caused orexacerbated by any type of gram-positive bacteria. In a preferredembodiment, crystalline or crystal-like daptomycin, daptomycin-relatedlipopeptide or other lipopeptide, or pharmaceutical compositionsthereof, are administered to a patient according to the methods of thisinvention. In another embodiment, the bacterial infection may be causedor exacerbated by bacteria including, but not limited to,methicillin-susceptible and methicillin-resistant staphylococci(including Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcussaprophyticus, and coagulase-negative staphylococci), glycopeptideintermediary-susceptible Staphylococcus aureus (GISA),penicillin-susceptible and penicillin-resistant streptococci (includingStreptococcus pneumoniae, Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus avium, Streptococcus bovis, Streptococcuslactis, Streptococcus sangius and Streptococci Group C, StreptococciGroup G and viridans streptococci), enterococci (includingvancomycin-susceptible and vancomycin-resistant strains such asEnterococcus faecalis and Enterococcus faecium), Clostridium difficile,Clostridium clostridiiforme, Clostridium innocuum, Clostridiumperfringens, Clostridium ramosum, Haemophilus influenzae, Listeriamonocytogenes, Corynebacterium jeikeium, Bifidobacterium spp.,Eubacterium aerofaciens, Eubacterium lentum, Lactobacillus acidophilus,Lactobacillus casei, Lactobacilllus plantarum, Lactococcus spp.,Leuconostoc spp., Pediococcus, Peptostreptococcus anaerobius,Peptostreptococcus asaccarolyticus, Peptostreptococcus magnus,Peptostreptococcus micros, Peptostreptococcus prevotii,Peptostreptococcus productus, Propionibacterium acnes, and Actinomycesspp.

The antibacterial activity of daptomycin against classically “resistant”strains is comparable to that against classically “susceptible” strainsin in vitro experiments. In addition, the minimum inhibitoryconcentration (MIC) value for daptomycin against susceptible strains istypically 4-fold lower than that of vancomycin. Thus, in a preferredembodiment, a compound of the invention, or a pharmaceutical compositionof any one of these crystalline or crystal-like lipopeptides, isadministered according to the methods of this invention to a patient whoexhibits a bacterial infection that is resistant to other antibiotics,including vancomycin. In addition, unlike glycopeptide antibiotics,daptomycin exhibits rapid, concentration-dependent bactericidal activityagainst gram-positive organisms. Thus, in a preferred embodiment,compounds of the invention, or a pharmaceutical composition of any oneof these crystalline or crystal-like lipopeptides, is administeredaccording to the methods of this invention to a patient in need ofrapidly acting antibiotic therapy.

The method of the instant invention may be used for a gram-positivebacterial infection of any organ or tissue in the body. These organs ortissue include, without limitation, skeletal muscle, skin, bloodstream,kidneys, heart, lung and bone. The method of the invention may be usedto treat, without limitation, skin and soft tissue infections,bacteremia and urinary tract infections. The method of the invention maybe used to treat community acquired respiratory infections, including,without limitation, otitis media, sinusitis, chronic bronchitis andpneumonia, including pneumonia caused by drug-resistant Streptoococcuspneumoniae or Haemophilus influenzae. The method of the invention alsomay be used to treat mixed infections that comprise different types ofgram-positive bacteria, including aerobic, caprophilic or anaerobicbacteria. These types of infections include intra-abdominal infections,pneumonia, bone and joint infections and obstetrical/gynecologicalinfections. The method of the invention also may be used to treat aninfection including, without limitation, endocarditis, nephritis, septicarthritis and osteomyelitis. In a preferred embodiment, any of theabove-described diseases may be treated using crystalline orcrystal-like daptomycin, daptomycin-related lipopeptide, antibacteriallipopeptide, or pharmaceutical compositions of any one of thesecrystalline or crystal-like lipopeptides.

Crystalline or crystal-like daptomycin, daptomycin-related lipopeptideor other lipopeptide may also be administered in the diet or feed of apatient or animal. If administered as part of a total dietary intake,the amount of daptomycin or other lipopeptide can be less than 1% byweight of the diet and preferably no more than 0.5% by weight. The dietfor animals can be normal foodstuffs to which daptomycin or otherlipopeptide can be added or it can be added to a premix.

The method of the instant invention may also be practiced whileconcurrently administering another form of daptomycin or otherlipopeptide antibiotic, e.g., one that is not crystalline orcrystal-like, or with one or more antifungal agents and/or one or moreantibiotics other than crystalline or crystal-like daptomycin or othercrystalline or crystal-like lipopeptide antibiotics. Co-administrationof an antifungal agent and an antibiotic other than crystalline orcrystal-like daptomycin or another lipopeptide antibiotic may be usefulfor mixed infections such as those caused by different types ofgram-positive bacteria, or those that caused by both bacteria andfungus. Furthermore, crystalline or crystal-like daptomycin or otherlipopeptide antibiotic may improve the toxicity profile of one or moreco-administered antibiotics. It has been shown that administration ofdaptomycin and an aminoglycoside may ameliorate renal toxicity caused bythe aminoglycoside. In a preferred embodiment, an antibiotic and/orantifungal agent may be administered concurrently with a compound ofthis invention, or in a pharmaceutical composition comprising a compoundof this invention.

Antibacterial agents and classes thereof that may be co administeredwith a compound of the present invention include, without limitation,penicillins and related drugs, carbapenems, cephalosporins and relateddrugs, aminoglycosides, bacitracin, gramicidin, mupirocin,chloramphenicol, thiamphenicol, fusidate sodium, lincomycin,clindamycin, macrolides, novobiocin, polymyxins, rifamycins,spectinomycin, tetracyclines, vancomycin, teicoplanin, streptogramins,anti-folate agents including sulfonamides, trimethoprim and itscombinations and pyrimethamine, synthetic antibacterials includingnitrofurans, methenamine mandelate and methenamine hippurate,nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol,pyrazinamide, para-amino salicylic acid (PAS), cycloserine, capreomycin,ethionamide, prothionamide, thiacetazone, viomycin, everninomycin,glycopeptide, glycylcylcline, ketolides, oxazolidinone; imipenen,amikacin, netilmicin, fosfomycin, gentamicin, ceftriaxone, Ziracin, LY333328, CL 331002, HMR 3647, Linezolid, Synercid, Aztreonam, andMetronidazole, Epiroprim, OCA_(—)983, GV_(—)143253, Sanfetrinem sodium,CS_(—)834, Biapenem, A_(—)99058.1, A_(—)165600, A_(—)179796, KA 159,Dynemicin A, DX8739, DU 6681; Cefluprenam, ER 35786, Cefoselis,Sanfetrinem celexetil, HGP_(—)31, Cefpirome, HMR_(—)3647, RU_(—)59863,Mersacidin, KP 736, Rifalazil; Kosan, AM 1732, MEN 10700, Lenapenem, BO2502A, NE_(—)1530, PR 39, K130, OPC 20000, OPC 2045, Veneprim, PD138312, PD 140248, CP 111905, Sulopenem, ritipenam acoxyl,RO_(—)65_(—)5788, Cyclothialidine, Sch_(—)40832, SEP_(—)132613,micacocidin A, SB_(—)275833, SR_(—)15402, SUN A0026, TOC 39, carumonam,Cefozopran, Cefetamet pivoxil, and T 3811.

In a preferred embodiment, antibacterial agents that may be coadministered with a compound according to this invention include,without limitation, imipenen, amikacin, netilmicin, fosfomycin,gentamicin, ceftriaxone, teicoplanin, Ziracin, LY 333328, CL 331002, HMR3647, Linezolid, Synercid, Aztreonam, and Metronidazole.

Antifungal agents that may be co administered with a compound accordingto this invention include, without limitation, Caspofungen,Voriconazole, Sertaconazole, IB_(—)367, FK_(—)463, LY_(—)303366,Sch_(—)56592, Sitafloxacin, DB_(—)289 polyenes, such as Amphotericin,Nystatin, Primaricin; azoles, such as Fluconazole, Itraconazole, andKetoconazole; allylamines, such as Naftifine and Terbinafine; andanti-metabolites such as Flucytosine. Other antifungal agents includewithout limitation, those disclosed in Fostel et al., Drug DiscoveryToday 5:25_(—)32 (2000), herein incorporated by reference. Fostel et al.disclose antifungal compounds including Corynecandin, Mer_WF3010,Fusacandins, Artrichitin/LL 15G256(, Sordarins, Cispentacin,Azoxybacillin, Aureobasidin and Khafrefungin.

Compounds of this invention, or a pharmaceutical composition of any oneor more of these crystalline or crystal-like lipopeptides, may beadministered according to this method until the bacterial infection iseradicated or reduced. In one embodiment, the crystalline orcrystal-like lipopeptide is administered for a period of time fromapproximately 3 days to approximately 6 months. In a preferredembodiment, the crystalline or crystal-like lipopeptide is administeredfor 7 to 56 days. In a more preferred embodiment, the crystalline orcrystal-like lipopeptide is administered for 7 to 28 days. In an evenmore preferred embodiment, the crystalline or crystal-like lipopeptideis administered for 7 to 14 days. The crystalline or crystal-likelipopeptide may be administered for a longer or shorter time period ifit is so desired. In a preferred embodiment, the lipopeptide isdaptomycin or daptomycin-related lipopeptide.

In order that this invention may be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

Example 1

Daptomycin was prepared by conventional techniques. The daptomycinpreparation was a pale yellow amorphous powder, with a solubility at 25°C. of greater than 1 g/mL in water and a solubility of 2.8 mg/mL inethanol. The amorphous daptomycin preparation was hygroscopic anddecomposed at 215° C.

The remaining examples describe crystallizing or precipitatinglipopeptides in the presence or absence of an organic precipitant (e.g.,PEG).

Example 2

In a microbatch crystallization, 25 μL of a daptomycin stock (20 mg/mLin methanol) was sequentially mixed with 15 μL of reagent stock (200 mMcalcium acetate, 0.1 M cacodylate (pH 6.5), 18% [w/v] PEG 8000 and 15 μLethylene glycol) to give a solution that was 27.5% aqueous component,45% methanol and 27.5% ethylene glycol. Urchin-like crystals were formedat a yield of 50% with a purity of 98% as measured by HPLC.

Example 3

A daptomycin stock was prepared by dissolving 440 mg daptomycin in 1 mLof a buffer containing 25 mM sodium acetate (pH 5.0) and 5 mM CaCl₂.Crystallization was done by the vapor diffusion (hanging drop) method,in which 5 μL of the daptomycin stock was added to 5 μL of 0.1 Mtri-sodium citrate dihydrate (pH 5.6), and 35% [v/v] tert-butanol inwater to form a drop. The drop was suspended over a reservoir solution(0.1 M tri-sodium citrate dihydrate (pH 5.6), and 35% [v/v] tert-butanolin water) in an air-tight environment until crystallization occurred.This method yielded urchin-like daptomycin crystals. See, e.g., FIG. 2.

Example 4

5 μL of a daptomycin stock prepared as in Example 3 was added to 5 μL ofa solution containing 0.1 M sodium cacodylate (pH 6.5), 0.2 M calciumacetate and 9% [w/v] PEG 8000. Crystallization was done by the vapordiffusion method as described in Example 3. This method yieldedneedle-like daptomycin crystals. See, e.g., FIG. 3.

Example 5

5 μL of a daptomycin stock prepared as in Example 3 was added to 5 μL ofa solution of 0.1 M sodium cacodylate (pH 6.5), 0.2 M zinc acetate and9% [w/v] PEG 8000 containing 0.1 μL benzamidine to give a finalconcentration of 220 mg/mL daptomycin. Crystallization was done by thevapor diffusion method as described in Example 3. This method yieldedrod-like daptomycin crystals. See, e.g., FIG. 4.

Example 6

One mL of daptomycin (97.1% pure as determined by HPLC) at aconcentration of 20-25 mg/mL in water was sequentially mixed with 231 μLwater, 77 μL of calcium acetate (pH 6.0), 960 μL propylene glycol and231 μL of 50% [w/v] PEG 4000. The solution was allowed to sit for 4-5hours at 4° C. Urchin-like crystals were formed at a yield of 75%. Thecrystalline daptomycin was washed with isopropanol. The daptomycin was98.4% pure as determined by HPLC.

Example 7

Daptomycin (200 mg, 97.1% pure) was dissolved in 2.54 mL water. Thedaptomycin solution was sequentially mixed in order with 10.0 mLmethanol, 0.78 mL 1 M calcium acetate (pH 6.0), 9.50 mL propylene glycoland 2.20 mL 50% [w/v] PEG 4000 to give a final volume of 25.02 mL. Themixture was tumbled at room temperature for 10-14 hours in a hematologymixer (Fischer). Crystals began to appear within a few hours. Finalyield was approximately 70-80% after 14 hours. The crystals wereharvested by centrifugation at 1000 rpm for 15 minutes. The supernatantwas removed and the crystals were resuspended in 12.5 mL isopropanol.The daptomycin suspension was transferred to a column (Biorad) and theisopropanol was removed by allowing it to drip by gravity. The crystalswere dried by a nitrogen stream. Any lumps were broken up during thedrying procedure to obtain a uniform dry sample. Crystals prepared bythis method were urchin-like and had a purity of 98.37%.

Example 8

Daptomycin was crystallized according to Example 7 except that PEG 8000was used in replacement of PEG 4000. The quantities of reagents used areidentical to those in Example 7. Crystals prepared by this method wereurchin-like and had a purity of 98.84%.

Example 9

Two daptomycin samples prepared according to Example 7 and one amorphoussample were analyzed for crystallinity using the USP <695> crystallinitytest. Daptomycin particles were mounted in mineral oil on a glass slideand then were examined by polarizing light microscope (PLM). Theparticles were determined to be crystalline if they were birefringent(have interference colors) and had extinction positions when the stagewas rotated.

The amorphous daptomycin sample consisted of lacy, flaky particles thatwere not birefringent. There were a few sliver-like areas in some of theflakes that had weak birefringence, but the particles were primarilyamorphous. In contrast, the daptomycin samples prepared according toExample 7 consisted of polycrystalline particles with weak birefringenceand some extinction, indicating that they were primarily crystalline.See FIG. 5.

Example 10

Two daptomycin samples prepared according to Example 7 and one amorphoussample were analyzed for crystallinity by x-ray powder diffraction. Thesamples were analyzed on a Siemens D500 Automated Powder Diffractometer(ORS 1D No. LD-301-4), which was operated according to ORS StandardOperation Procedure EQ-27 Rev. 9. The diffractometer was equipped with agraphite monochromator and a Cu (λ=1.54 Å) x-ray source operated at 50kV, 40 mA. Two-theta (0) calibration is performed using an NBS micastandard (SRM675). The samples were analyzed using the followinginstrument parameters:

Measuring Range for 2 Θ (degrees) 4.0-40.0 Step Width (degrees) 0.05Measuring Time per Step (secs) 1.2  Beam Slits 1(1°), 2(1°), 3(1°),4(0.15°), 5(0.15°).

Sample preparation was performed according to ORS Standard OperationProcedure MIC-7 Rev. 1 using a zero background sample plate.

All samples were done using a Cu (λ=1.54 Å) x-ray source. The amorphousdaptomycin sample did not show any peaks by x-ray powder diffraction.See FIG. 6. In contrast, the two daptomycin samples both showed peaks byx-ray powder diffraction. The diffraction angle (2 θ) of the firstdaptomycin sample (FIG. 7) was 19.225, 23.242, 23.427 and 23.603(degree). The diffraction angle (2 θ) for the second daptomycin sample(FIG. 8) was 10.966, 19.205 and 23.344 (degree). The first crystallinedaptomycin sample also showed a small peak between 10-11°. See FIG. 7.

Example 11

Daptomycin was dissolved in water. Sodium acetate was added to achieve afinal concentration of 187 mM. Calcium chloride was added to achieve afinal concentration of 28 mM. The daptomycin solution was mixed andisopropanol was added to a final concentration of 78.4%. The solutionwas mixed and incubated. A precipitated material was formed afterincubation. The precipitated material appeared to be urchin-likecrystals of approximately 60 μm diameter by optical microscopy. Thematerial was then dried. The dry material contained approximately 30-40%salt. After drying, powder x-ray diffraction was performed. The powderx-ray diffraction did not show the presence of crystals in the drieddaptomycin precipitate.

Example 12

One gram of daptomycin (approximately 91.5% purity as measured by HPLC)was added to 16.8 mL of distilled water and dissolved. 2.5 mL of 1Mcalcium acetate (pH 6.1) and 60 mL of isopropanol was added. Thesolution was placed in a 27° C. water bath and permitted to equilibrateto temperature of the water bath. 5 mL aliquots of isopropanol wereslowly added until the solution became cloudy (a total of approximately30 mL isopropanol). The solution was incubated overnight at 27° C. toform a precipitate. The precipitate appeared to contain urchin-likecrystals of approximately 60 μm by optical microscopy. See FIG. 2.

The daptomycin precipitate was poured into a pressure filter/dryingfunnel and filtered by gravity. The precipitate was washed twice with 25mL each time of a washing solution (80% isopropanol and 20% solution Awhere solution A consists of 18 mL of water and 2 mL of glacial aceticacid) and allowed to drip by gravity overnight. The precipitate was thentransferred to a desiccator and dried under vacuum. After drying, powderx-ray diffraction was performed. The powder x-ray diffraction did notshow the presence of crystals in the dried daptomycin precipitate.However, purity analysis of the precipitated material by HPLC showedthat the material was 98.2% pure daptomycin. Significantly, thedaptomycin preparation after precipitation has significantly lessanhydro-daptomycin than the daptomycin preparation before precipitation.

Without wishing to be bound by any theory, applicants believe that theconditions used to precipitate the daptomycin in Examples 11 and 12actually produce a crystalline form of daptomycin but that thesubsequent washing steps and/or drying steps cause the crystallinedaptomycin to revert to a non-crystalline form. Nonetheless, thenon-crystalline daptomycin is still crystal-like as shown in FIG. 3 bythe birefringence of a crystal sample in polarized light.

Example 13

A fermentation culture of S. roseosporus NRRL Strain 15998 is conductedin a controlled decanoic acid feed fermentation at levels that optimizethe production of the antibiotic while minimizing the production ofcontaminants. The residual decanoic acid feed is measured by gaschromatography and the target residual level is 10 ppm decanoic acidfrom the start of induction (approximately at hour 30) until harvest.Centrifugation of the culture and subsequent analysis of the clarifiedbroth are used to measure the production of daptomycin by HPLC. Theharvest titer is typically between 1.0 and 3.0 grams per liter offermentation broth.

The fermentation culture is harvested either by microfiltration using aPall-Sep or equivalent microfiltration system, or by fullcommercial-scale centrifugation and depth filter. The clarified broth isapplied to an anion exchange resin, Mitsubishi FP-DA 13, washed with of30 mM NaCl at pH 6.5 and eluted with of 300 mM NaCl at pH 6.0-6.5.Alternatively, the FP-DA 13 column is washed with of 30 mM NaCl at pH6.5 and eluted with of 300 mM NaCl at pH 6.0-6.5. The pH is adjusted to3.0-4.8 and the temperature is adjusted to 2-15° C. Under theseconditions, daptomycin forms a micelle. The micellar daptomycin solutionis filtered-washed using a 10,000 NMW ultrafilter (AG Technology Corp.UF hollow fiber or equivalent) in any configuration. The daptomycinmicelles are retained by the filter, but a large number of impuritiesare eliminated because they pass through the 10,000 NMW filter.Ultrafiltration of daptomycin micelles increases daptomycin purity toapproximately 80-90%.

The daptomycin preparation is then crystallized or precipitated understerile conditions using one of the methods described above. In apreferred embodiment, the daptomycin is crystallized or precipitatedaccording to the protocol described in Examples 7, 8 or 12 except thatit can be scaled up for large preparation of daptomycin. The crystallineor crystal-like daptomycin is separated from thecrystallization/precipitation solution by filtration, preferably byvacuum filtration. The crystalline or crystal-like daptomycin is washedwith washing solution (see Example 3). The crystalline or crystal-likedaptomycin is then vacuum dried under sterile conditions using a 0.65 m³Klein Hastelloy-B double cone vacuum dryer or equivalent apparatus.Vials are then filled with either 250 or 500 mg of dried crystallinedaptomycin per vial. FIG. 9 shows a flowchart of this manufacturingmethod.

Example 14

Fermentation of S. roseosporus, microfiltration of the fermentationculture and anion exchange chromatography is performed as described inExample 13. The daptomycin preparation is approximately 35-40% pure atthis point. After anion exchange chromatography, the daptomycin iscrystallized or precipitated according to the protocol described inExample 13. The daptomycin is then washed and dried according to theprotocol set forth in Example 13. The dried crystalline or crystal-likedaptomycin is then used to fill sterile vials as described in Example13. FIG. 6 shows a flowchart of this manufacturing method.

Example 15

Fermentation of S. roseosporus and microfiltration of the fermentationculture is performed as described in Example 13. After microfiltration,the fermentation culture is subjected to size exclusion ultrafiltrationas described in Example 13. The daptomycin preparation is approximately35-40% pure at this point. After ultrafiltration, the daptomycin iscrystallized or precipitated according to the protocol described inExample 13. The daptomycin is then washed and dried according to theprotocol set forth in Example 13. The dried crystalline or crystal-likedaptomycin is then used to fill sterile vials as described in Example13. FIG. 7 shows a flowchart of this manufacturing method.

Example 16

Fermentation of S. roseosporus and microfiltration of the fermentationculture is performed as described in Example 13. The daptomycinpreparation is 5-10% pure at this point. After microfiltration, thefermentation culture is crystallized or precipitated according to theprotocol described in Example 13. The daptomycin is then washed anddried and used to fill sterile vials as described in Example 13. FIG. 8shows a flowchart of this manufacturing method.

Example 17

CB-131547 (see Figure x), a cyclic lipopeptide analog of daptomycin, wasprepared via a semi-synthesis route from daptomycin. The CB-131547 was apale yellow amorphous powder, with a solubility at 25° C. of ˜80 mg/mLin normal saline.

CB-131547 (60 mg, ˜90% pure) is dissolved in 2.5 mL water. The CB-131547solution is sequentially mixed in order with 5.0 mL methanol, 0.2 mL 1 Mcalcium acetate (pH 6.0), 2.5 mL propylene glycol, and 1.0 mL 50% (w/v)PEG 4000 to give a final volume of 11.2 mL. The solution is allowed tosit for 4 to 24 hours at 4° C. CB-131547 crystals are formed at a yieldof ˜70% with a purity ˜98.0% as determined by HPLC.

Example 18

CB-131547 (see Figure x), a cyclic lipopeptide analog of daptomycin, wasprepared via a semi-synthesis route from daptomycin. The CB-131547 was apale yellow amorphous powder, with a solubility at 25° C. of ˜80 mg/mLin normal saline.

CB-131547 (60 mg, ˜90% pure) is dissolved in 2.5 mL water. 0.2 mL 1 Mcalcium acetate (pH 6.0) and 8 mL of isopropanol is added. The solutionis allowed to equilibrate at room temperature (25° C.) for 5 minutes.One mL aliquots of isopropanol are slowly added until the solutionbecomes cloudy. The solution is stored at room temperature overnight toform crystals.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1-56. (canceled)
 57. A daptomycin calcium salt produced by a methodcomprising the steps of: a) combining daptomycin in a solutioncomprising an alcohol selected from the group consisting of isopropanol,tert-butanol, and methanol, and calcium acetate at a pH of about 5.5 to7.5 and a temperature of about 0 to 30 degrees C.; b) allowing thedaptomycin to precipitate or crystallize from the solution; and c)obtaining the daptomycin calcium salt from the precipitated orcrystallized daptomycin.
 58. The daptomycin calcium salt of claim 57,wherein the solution comprises isopropanol and the temperature of thesolution is about 20 to 30 degrees C.
 59. The daptomycin calcium salt ofclaim 57, wherein the solution comprises isopropanol and the daptomycinis precipitated or crystallized at a pH of about 5.9 to 6.3.
 60. Thedaptomycin calcium salt of claim 57, wherein the daptomycin of step a)is produced by a method comprising the steps of: i) fermentingStreptomyces roseosporus to obtain a fermentation culture containingdaptomycin; ii) clarifying the daptomycin from the fermentation cultureto obtain a daptomycin preparation; iii) subjecting the daptomycinpreparation to size exclusion ultrafiltration to obtain a filtereddaptomycin preparation; iv) subjecting the filtered daptomycinpreparation to hydrophobic interaction chromatography; v) subjecting thedaptomycin from the hydrophobic interaction chromatography to anionicexchange chromatography; vi) adjusting the daptomycin obtained from theanion exchange chromatography to about pH 3.0-4.8 and about 2-15° C.;vii) filtering the daptomycin of step vi) under conditions in which thedaptomycin is retained on the filter; and viii) obtaining the daptomycinof step a) from the daptomycin of step vii).
 61. The daptomycin calciumsalt of claim 57 having a daptomycin purity of at least 93%.
 62. Thedaptomycin calcium salt of claim 57, wherein the daptomycin combined inthe solution of step a) has a daptomycin purity of less than about 93%.63. The daptomycin calcium salt of claim 57 having a daptomycin purityof at least about 93%, wherein the daptomycin combined in the solutionof step a) has a daptomycin purity of less than 93%.
 64. A daptomycincalcium salt produced by a method comprising the steps of: a) forming adaptomycin preparation comprising a solution of daptomycin at a pH ofabout 3.0-4.8 and a temperature of about 2-15° C., b) filtering thedaptomycin preparation under conditions in which at least a portion ofthe daptomycin is retained on the filter; c) forming a solutioncomprising the retained daptomycin of step b), isopropanol and calciumacetate; d) maintaining the retained daptomycin from the solution instep c) at a temperature of about 0 to 30 degrees C. and a pH of about5.5 to 7.5 to form a precipitated or crystalline daptomycin calciumsalt; and e) collecting the precipitated or crystalline daptomycincalcium salt.
 65. The daptomycin calcium salt of claim 64, wherein thedaptomycin in the daptomycin preparation of step a) is obtained by aprocess comprising the step of i) fermenting Streptomyces roseosporus toobtain a fermentation culture containing daptomycin.
 66. The daptomycincalcium salt of claim 65, wherein the daptomycin in the daptomycinpreparation of step a) is obtained by a process further comprising thesteps of: ii) clarifying the daptomycin from the fermentation culture toobtain a daptomycin preparation; and iii) subjecting the clarifieddaptomycin preparation from step i) to at least one of hydrophobicinteraction chromatography and anion exchange chromatography to obtainthe daptomycin for the daptomycin preparation of step a).
 67. Thedaptomycin calcium salt of claim 64, wherein the daptomycin from thesolution in step c) is maintained at a temperature of about 20 to 30degrees C. and a pH of about 5.9 to 6.3.
 68. A daptomycin calcium saltproduced by a method comprising the steps of: a) fermenting Streptomycesroseosporus to obtain a fermentation culture containing daptomycin; b)clarifying the daptomycin from the fermentation culture to obtain adaptomycin preparation; c) subjecting the daptomycin preparation to sizeexclusion ultrafiltration to obtain a filtered daptomycin preparation;d) subjecting the filtered daptomycin preparation to hydrophobicinteraction chromatography; e) subjecting the daptomycin obtained fromthe hydrophobic interaction chromatography to anion exchangechromatography; f) altering at least one of the pH, temperature and saltconcentration of the daptomycin obtained from the anion exchangechromatography; g) filtering the daptomycin of step f) under conditionsin which the daptomycin is retained on the filter; and h) forming asolution containing the daptomycin of step g), isopropanol, and calciumacetate; i) precipitating or crystallizing daptomycin from the solutionat a temperature of about 0 to 30 degrees C. and a pH of about 5.5 to7.5; and j) collecting the precipitated or crystallized calcium salt ofdaptomycin from the solution.
 69. The daptomycin calcium salt of claim68, wherein the daptomycin is precipitated or crystallized from thesolution at a temperature of about 20 to 30 degrees C. and a pH of about5.9 to 6.3.
 70. A composition comprising a daptomycin calcium salthaving a purity of at least about 98%, the daptomycin calcium saltobtained by a process comprising the steps of: a) combining daptomycinin a solution comprising an alcohol selected from the group consistingof isopropanol, tert-butanol, and methanol, and a salt selected from thegroup consisting of calcium acetate and calcium chloride at a pH ofabout 5.5 to 7.5 and a temperature of about 0 to 30 degrees C.; b)allowing the daptomycin to precipitate or crystallize from the solution;c) obtaining daptomycin calcium salt from the precipitated orcrystallized daptomycin.
 71. The composition of claim 70, wherein thecomposition comprises a lower level of anhydro-daptomycin and/or theβ-isomer of daptomycin compared to the daptomycin combined in thesolution of step a).
 72. The composition of claim 70, wherein thedaptomycin of step a) is obtained by a process comprising the steps of:i) fermenting Streptomyces roseosporus to obtain a fermentation culturecontaining daptomycin; ii) clarifying the fermentation culture to obtaina clarified daptomycin preparation; and iii) subjecting the clarifieddaptomycin preparation to anion exchange chromatography.
 73. Thecomposition of claim 70, wherein the salt is calcium acetate.
 74. Thecomposition of claim 70, wherein the temperature of the solution isabout 2-15 degrees C.